This invention relates to a novel bacteria strain of Pseudomonas fluorescens designated as ATCC 55939 and a method for large scale screening of native rhizosphere microflora, to identify and characterize naturally occurring rhizosphere-competent biocontrol bacteria from non-sterilized soil, which could effectively colonize plant roots. The present invention relates more specifically to the use of a novel strain of Pseudomonas fluorescens as a biocontrol agent for controlling plant fungal disease particularly those diseases caused by fungus of the genus Fusarium sp., Rhizoctonia sp. and Pythium sp.
The fungal pathogens play a major role in the development of diseases on many important field and horiculture crops which often results in the poor plant yields. Considering the cost of chemical pesticides and hazard involved, biological control of plant diseases is now increasingly capturing the imagination of plant microbiologists. Frequent failure of the added microorganisms to become established is not surprising because the biological associations and antagonisms within the ecosystem determine the composition of the microflora, the climax population being a reflection of the physical and chemical characteristics of the habitat.
A major factor in the unsuccessful commercialization of rhizosphere bacteria has been the inconsistency of field test results. Reasons for the reported variability include nonpersistence on seed before it is planted and poor bacterial establishment on seed and roots, please refer Burr, T, J., and A. Caesar, Crit. Rev. Plant Sci. 2: 1-20 (1984); Gaskins, M. H. et al. Ecosystems Environ. 12: 99-116 (1985); Liang, L. et al. Appl. Environ. Microbiol. 44: 708-714 (1982); O""Sullivan, D. J., and F. O""Gara, Micrbiol. Rev. 56: 662-676 (1992); Schrotk, M. N., and J. G. Hancock. Disease suppressive soil and root colonizing bacteria. Science 216: 1376-1381 (1981); Weller, D. M., Ann. Rev. Plant Pathol. 26: 379-407 (1988). The introduced microorganism must colonize plant roots and demonstrate rhizosphere competence before its further utilization as biological control and/or, plant growth promoting agent. When the proper bacterial strain is used, plant roots are extensively colonized by the introduced strain, which suggests a close bacteria-plant association that allows for beneficial plant growth or disease protection, see for example, Schmidt, E. L., Ann. Rev. Microbiol. 33: 355-376 (1979).
The isolation and development of plant beneficial bacteria applicable to a varietv of crops. soils. and locations will depend on the development of improved detection and screening procedures that more rapidly identify beneficial bacteria. While prior methods have been somewhat effective, such methods have had inherent shortcomings. For example, the current methods which are required to ascertain bacterial root colonization capacity are laborious and often produce highly variable results. Bennett and Lynch J. Gen. Microbiol. 125: 95-102 (1981), developed a closed test tube assay for measuring root colonization capacity of bacteria under gnotobiotic conditions which proved useful for studying specific microbial interactions in the rhizosphere. However, it is not possible to extrapolate results obtained in sterilized soils to those expected under field conditions, see for example, Klopper, J. W. Plant growth-promoting rhizobacteria and plant growth under gnotibiotic conditions. Phytopathology. 71: 642-644 (1981). Scher, F. M. et al. Can. J. Microbiol. 30: 151-157 (1984) measured the root colonization capacity of bacteria on maize in raw soil-sand closed test tube assay and demonstrated that root population densities determined in the soil-sand assay were comparable with those determined with plants grown in soils under greenhouse conditions. However, Scher et al. did not compare the competitive fitness of Rif mutants with the wild type strain. Compeau, G. et al. Appl. Environ. Microbiol. 54: 2432-2438 (1988) have demonstrated that colonization of soil by a species which is isogenic to a challenging organism may preempt the colonization of the soil by the second organism. This is true even when organisms display identical fitness. This interaction may be important in the failure of introduced strains to increase in number when introduced into their own environment. Thus, it is desirable to obtain novel strains of biocontrol agents which effectively control the growth of plant pathogens, particularly fungi, and are able to aggressively compete with indigenous bacteria and other microflora that exist in the rhizosphere of the plant.
This invention relates to a simple sand-live soil assay method, for large scale screening of the rhizosphere-competent bacteria that are effective in suppressing plant pathogens, that has been developed. Screening for chickpea rhizosphere competitive bacteria having biological control property was conducted at three different stages: development of screening method for large scale initial selection of bacteria isolates from chickpea rhizosphere, testing of biocontrol activity under in vitro conditions and screening of antibiotic resistant mutants for rhizosphere competence in nonsterile field soil, which assay has disclosed one Pseudomonas fluorenscens NBRI 1303 (ATCC 55939) that is effective in suppressing plant pathogens, including Fusarium oxysporum f. sp. ciceri, Rhizoctonia bataticola and Pythium sp. in chickpeas. The purified bacterial strain can be used as an active agent for biocontrol compositions and can also be used for enhancement of chickpea plant growth and yield, as well as for the production of antibiotics directed against phytopathogenic fungal diseases.
Accordingly, it is an object of the present invention to provide a method of raw (non-sterile) soil assay for large scale screening of native rhizosphere microflora of chickpeas that identifies and characterizes naturally occurring rhizosphere bacteria that effectively colonize chickpea roots.
Another object of the present invention is to provide a biocontrol agent that is useful in methods for suppression of fungal infection in chickpeas and thereby enhances plant yields.
Still another object of the present invention is the use of a biological control agent(s) that produces one or more metabolites capable of inhibiting fungal pathogens of chickpeas or other plants. The use of such a biological control agent instead of seed treatment and soil treatment chemical fungicides allows reduction of environmental contamination.
Yet another object of the present invention is to provide a method for biocontrol of pathogenic fungi F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp. in plants by application of an isolated P. fluorenscens strain.
Other objects and advantages of the invention will become apparent from the ensuing description.
Accordingly, the present invention provides a method for large scale screening of native rhizospheric competent bacteria for strains that will control plant pathogens thereby promoting plant growth in field grown chickpea crops, said method comprising:
a. harvesting chickpea crops from a disease-suppressive field after five months of plant growth;
b. subjecting the said harvested crop roots to drying in the field for a post harvest period of four weeks;
c. isolating bacterial strains from the said chickpea roots by standard methods;
d. selecting rifampin resistant bacterial strains showing growth comparable to wild type on agar plates containing 100 (xcexcg rifampin/ml);
e. evaluating the root colonization capability of the bacterial strain obtained in step (d) in the greenhouse on the basis of average root colonization values of rifampin resistance strains after four weeks of post planting period;
f. selecting bacterial strains from the strains evaluated in step (e) with 107-108 CFU/g root; and
g. screening a novel strain from the bacterial strains obtained in step (f) for its invitro inhibition against F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp. to produce a biologically pure culture of bacteria that suppresses diseases caused by pathogens in chickpea crops as determined by passing the screen test of the invention.
Basically, the invention provides a biologically pure culture of the bacteria of the invention wherein said culture of bacteria comprises a strain of Pseudomonas fluorenscens. The culture of bacteria comprises a strain of Pseudomonas fluorescens that has been deposited at ATCC and designated as ATCC 55939.
The invention also relates to a screening method wherein initial screening time is reduced in the present assay system by directly inoculating the rifampin resistant strains into seeds without checking for the stability of the mutation. The present invention also provides a screening method in which initial screening time is reduced as compared to the current assay system by directly inoculating the rifampin resistant strains into seeds without any taxonomic identification.
Another aspect of the invention is to provide a method wherein the novel bacterial strain isolated in the step (g) is used in greenhouse seed bacterization, providing 10 to 25% or more increase in germination of chickpea seedlings in the presence of phytopathogenic fungal disease conducive soil from the trial fields.
Yet another embodiment of the invention is to provide a method wherein the novel strain isolated in the step (g) is used in greenhouse seed bacterization, providing 35 to 60% increase in survival rate of chickpea seedlings in the presence of the phytopathogenic fungal disease conducive soil from the trial fields.
Further, the invention provides a method wherein the novel strain isolated in step (g) is used in greenhouse seed bacterization to provide more that 10 to 25% increase in dry weight of chickpea seedlings exposed to phytopathogenic fungal disease conducive soil from the trial fields. The novel strain isolated in step (g) is used in greenhouse seed bacterization to provide more than 15 to 25% increase in the length of chickpea shoots growing in the presence of phytopathogenic fungal disease conducive soil from the trial fields. The applicants also noticed that the novel strain isolated in step (g) provided 10-15% increase in shoot length in chickpea plants in fields naturally infested with F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp. In addition, the present novel strain of bacteria isolated in step (g) provided 15-20% increase in shoot dry weight in chickpea plants in fields that were naturally infested with F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp.
The invention further provides a method in which the novel strain of bacterium isolated in step (g) provided 20 to 30% increase in seed dry weight in chickpea plants in fields that were naturally infested with F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp. The present strain isolated in step (g) was used in greenhouse seed bacterization providing 15 to 25% increase in the length of chickpea roots in the presence of phytopathogenic fungal disease conducive soil from the trial fields.
Still another aspect of the invention is to provide a method wherein the novel strain isolated in step (g) provides rhizospheric competence for the full growing season of the chickpea plant. The applicants also observed that the novel bacteria strain isolated in step (g) exhibits the capability to solubilize phosphate in the presence of salt (NaCl) in a concentration range of 0 to 5% and has the capability to solubilize phosphate at a temperature in the range 30-45xc2x0 C. Further, it was observed that the present novel bacteria strain isolated in step (g) exhibits the capability of solubilizing phosphate at a pH in the range of 7-9.
The present invention relates to an improved method that requires less screening time for isolating and identifying naturally occurring rhizosphere-competent bacteria from non-sterilized soil. More specifically, the present invention relates to a novel P. fluorescens strain that can be used as a biocontrol agent for suppression of the pathogenic fungi F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp. in chickpeas.
Current available methods require a lot of time to screen the native microorganisms for rhizospheric competence. In the present study, the screening time, compared to previous reports (see for example, Compeau, G. et al Appl. Environ. Microbiol. 54: 2432-2438 (1988); Juhnke, M. E. et al Appl Environ. Microbiol. 53: 2793-2799 (1987); Lewis, D. M. et al. an. J. Microbiol. 33: 343-345 (1987); Liang, L. et al Appl. Environ. Microbiol. 44: 708-714 (1982); Loper, J. E. et al Appl. Environ. Microbiol. 49: 416-422 (1985); Scher, F. M. et al. Can. J. Microbiol. 30: 151-157 (1984) Turco, R. F. et al. Soil Biol. Biochem. 18: 259-262 (1986)), has been reduced in two respects. First, Rif strains in the present assay system were directly inoculated into seeds without any check for the stability of the mutation. This stability check usually requires at least 25 passes through non-selective media. It is anticipated that any Rif strain that shows high CFU/g root 30 days after inoculation should by able to do so only if it is capable of sustained growth while competing against the native microorganisms. Secondly, no attempts have been made to taxonomically identify all the strains.
Using this method, one chickpea rhizosphere competent strain was selected after screening four hundred and seventy eight bacteria, and taxonomically identified as Pseudomonas fluorenscens NBRI 1303 which is also deposited in ATCC and designated as ATCC 55939.
The results indicate that P. fluorescens NBRI 1303 is an aggressive chickpea rhizosphere colonizer and can survive in the field at temperatures in the range of 0xc2x0 C. to 55xc2x0 C. In addition, this bacterial strain appeas to produce one or more antifungal metabolites which inhibit the growth of pathogenic fungi F. oxysporum f. sp. ciceri, R, bataticola and Pythium sp. or other fungal pathogens of chickpeas since the culture supernatant exhibits growth inhibitory effects for pathogenic fungi F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp. Greenhouse test and field trial of P. fluorescens NBRI 1303 (ATCC 55939) demonstrated the usefulness of the strain as an inoculum for improved plant performance and therefore P. fluorenscens NBRI 1303 (ATCC 55939) may be used as a biocontrol agent.
The characteristics of the subject P. fluorescens NBRI 1303 (ATCC 55939) were determined according to the morphological and physiological descriptions provided in Bergy""s Manual of Determinative Bacteriology. The taxonomic characteristics of the strain Pseudomonas fluorescens are given in Table 1.
A representative isolate of the present invention has been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20110-2209, USA in accordance with the Budapest Treaty. This isolate is P. fluorescens strain NBRI 1303 assigned number ATCC 5939 deposited Feb. 21, 1997.
In addition to the other properties noted above, the novel strain P. fluorescens NBRI 1303 (ATCC 55939) also has the following qualities which are important for use as a biological control agent for chickpeas:
(i) being a naturally occurring isolate that does not require any genetic manipulations, to be effective
(ii) being easily cultured and developed for commercial purposes,
(iii) exhibiting capability to be rhizosphere competent for the full growing season,
(iv) being suppressive of one or more phytopathogenic fungi,
(v) exhibiting capability to soluble phosphorus,
(vi) enhancing the yield of the host plant,
(vii) being environment friendly, unlike chemical fungicides and fertilizers.
While the present method is described for evaluating rhizospheric colonization potential of a diverse group of naturally occurring bacteria isolated from non-sterilized soil from the rhizosphere of chickpeas as a host plant, P. fluorescens (organisms involved in promoting plant growth, suppression of plant disease, phosphate solubilization and phytoremediation), it should be recognized that the plants of the present invention may be any genus and/or species of plant or any genus and/or species of plant associated bacteria or combination thereof that will assist the plant growth through association with the plant root. These indigenous bacteria would then be available for use as biological control agents or plant growth-promoting bacteria applied as seed inoculants directly, and/or after genetic engineering.
The biocontrol agent or the substances produced by the biocontrol agent of the present invention may be used in any manner known in the art, including coating seeds in the presence or absence of carrier or direct seedling or soil treatment. While certain detailed and preferred embodiments have been disclosed for biological control of Fusarium oxysporum f. sp. ciceri, Rhizoctonia bataticola and Pythium sp. in chickpeas, numerous variations, modifications and embodiments are possible, and accordingly; all such variations, modifications and embodiments are to be regarded as being within the spirit and scope of the present invention.